Methods for diabetes susceptibility assessment in asymptomatic patients

ABSTRACT

Assays for identifying human patients at risk for developing insulin-dependent diabetes mellitus rely on detection of autoantibodies to a 38 kD autoantigen present in pancreatic β-cells in patient sera. It has been found that autoantibodies to this particular autoantigen developed in patients well before clinical onset of the disease in a significant subpopulation of prediabetic patients. Useful assays will frequently combine detection of autoantibodies to the 38 kD autoantigen with detection of other known markers of IDDM, such as autoantibodies to a 64 kD autoantigen (glutamic acid decarboxylase).

This is a Continuation of application Ser. No. 08/346,313 filed Oct. 28,1994, U.S. Pat. No. 5,674,692, which is a continuation of applicationSer. No. 08/048,886 filed Apr. 16, 1993, abandoned, all of which areincorporated by reference in their entirety.

This application contains subject matter which is related to thatdisclosed in application Ser. No. 07/756,207, filed on Sep. 6, 1991,abandoned, and Ser. No. 07/984,935, filed Dec. 3,1992, abandoned, thefull disclosures of which are incorporated herein by reference.

This invention was made with government support under Grant No. DK41822-01-04 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for identifyingpatients who are at risk of developing insulin-dependent diabetesmellitus (IDDM). More particularly, the present invention relates to thedetection of an approximately 38 kD autoantigen associated with diabetesin serum of patients prior to clinical onset of the disease.

Insulin-dependent diabetes mellitus (IDDM) primarily afflicts youngpeople. Although insulin is available for treatment, the several foldincreased morbidity and mortality associated with this disease urge thedevelopment of early diagnostic and preventive methods. The destructionof pancreatic β-cells, which precedes the clinical onset of IDDM, ismediated by autoimmune mechanisms. Among the most thoroughly studiedautoimmune abnormalities associated with the disease is the highincidence of circulating β-cell specific autoantibodies at the time ofdiagnosis. Family studies have shown that the autoantibodies to certainβ-cell autoantigens appear prior to overt IDDM by a number of years,suggesting a long prodromal period of humoral autoimmunity beforeclinical symptoms emerge. The family studies have also documented aslow, progressive loss of insulin response to intravenous glucose in theyears preceding diagnosis. The presence of β-cell specificautoantibodies in the prediabetic period is likely to reflect theongoing autoimmune process, one that eventually leads to critical β-celldepletion and insulin dependency. It has been estimated that only 10% ofthe total β-cell mass remains at the time of clinical onset.

Thus, methods for early and accurate identification of susceptibleindividuals are needed. Assays that can detect autoantibodies associatedwith early humoral autoimmunity accompanying β-cell destruction areparticularly desirable. The classical method for detecting islet cellautoantibodies (ICA) is by immunohistology using frozen pancreaticsections. Family studies, however, have shown that the β-cellcytoplasmic antibodies measured by this method are of insufficientspecificity to serve as a single marker of susceptibility. Moreover, ICAare very difficult to standardize, and interpretation of the stainedsection is subject to observer bias. Thus, there has been no way todefine what is a “positive” specimen. More accurate assays may beachieved by employing more specific markers, either alone or incombination with ICCA. Alternative markers include autoantibodies to anapproximately 64 kD autoantigen, insulin autoantibodies, and MHC classII DR/DQβ haplotype.

The 64 kD autoantigen holds particular promise as a diagnostic markerfor IDDM. Autoantibodies to the 64 kD autoantigen have an incidence offrom about 70% to 80%. both at the time of clinical onset of the diseaseand the prediabetic period. The presence of the autoantibodies has beenshown to precede overt IDDM by several years and familial studies.Recently, inventors herein together with another research group havediscovered that the 64 kD autoantigen of pancreatic β-cells associatedwith IDDM is glutamic acid decarboxylase (GAD) (E.C. 4.1.1.15) which isan abundant protein of GABA-secreting neurons in the central nervoussystem (CNS). Based on this discovery, numerous conventional assayformats have become available for detecting autoantibodies to the 64 kDautoantigen (GAD) to permit patient screening.

Identification of autoantibodies to the 64 kD autoantigen, however, isinsufficient by itself as a screening test to identify patients at riskfor developing IDDM. As stated above, autoantibodies to the 64 kDautoantigen are present in only 70% to 80% of patients who later developIDDM, thus failing to identify a significant number of susceptibleindividuals. It would therefore be desirable to provide additional andalternative markers which are able to predict IDDM susceptibility in atleast a portion of those patients who do not develop antibody to the 64kD autoantigen. Such markers should be present at an early stage ofβ-cell destruction prior to the clinical onset of IDDM, and shouldremain detectable from that early stage through the time of clinicalonset. This marker should be identifiable in patient serum, thusfacilitating screening, and should preferably be compatible withdetection of autoantibodies to the 64 kD autoantigen.

2. Description of the Background Art

Antibodies to a 38 kD antigen present in human islets have been detectedby immunoprecipitation with sera from patients suffering from insulindependent diabetes mellitus. Baekkeskov et al. (1982) Nature 298:167-169and Aanstoot et al. (1991) Abstract 898, Diabetes 40 Supp. 1, page 225A.A 38 kD islet cell protein has been reported in the BB-rat, an animalmodel of insulin dependent diabetes mellitus. Ko et al. (1991)Diabetologia 34:548-554. T-cell reactivity to an insulinoma protein ofapproximately 38 kD has been detected in newly diagnosed diabeticpatients. Roep et al. (1990) Nature 345:632-634 and Lancet (1991)337:1439-1441.

Antibodies to the 65 kD isoform of the enzyme glutamic aciddecarboxylase (GAD₆₅) have been identified in 70% to 80% of individualsexperiencing β-cell destruction and development of insulin dependentdiabetes mellitus. Baekkeskov et al. (1990) Nature 347:151-156 and WO92/04632. Some patients having islet cell antibodies (ICA) identified byimmunofluorescence of frozen pancreas sections do not have antibodies toGAD₆₅. Seissler et al. (1991) Diabetologia 34:548-554.

SUMMARY OF THE INVENTION

The present invention comprises methods for assessing the risk ofdeveloping insulin-dependent diabetes mellitus (IDDM) in asymptomatichuman patients. The methods rely on detecting the presence ofautoantibodies to a 38 kD amphiphilic membrane-bound islet cell proteinhaving a pI in the range from 5.6 to 6.1. It has been found thatautoantibodies to the 38 kD autoantigen appear in a significant subgroupof patients who later develop IDDM, with the appearance occurring wellbefore clinical onset of the disease, usually more than one year priorto clinical onset, and often two years or more prior to clinical onset.It has been also found that autoantibodies to the 38 kD autoantigen arepresent in a significant number of patients who do not displayautoantibodies to the 64 kD autoantigen (GAD₆₅). Thus, assays accordingto the present invention will preferably rely on detection of antibodiesto both the 38 kD autoantigen and the 64 kD autoantigen (GAD₆₅), withthe presence of either autoantibody being predictive of IDDM.

A further understanding of the nature and advantages of the inventionwill become apparent by reference to the remaining portions of thespecification and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a fluorogram of an SDS-PAGE showing immunoprecipitation ofmembrane and cytosol fractions of ³⁵S-methionine labeled islet cellproteins with sera from newly diagnosed diabetic patients I₁-I₁₆ (lanes1-18), a stiff-man syndrome serum (lane 19) and sera from healthycontrols C1-C3 (lanes 20-22). GAD₆₅ which splits into two bands α and β,can be seen in immunoprecipitates from both membrane and cytosol.fractions with serum from patient I₁₆ (lanes 16 and 18) whereas the 38kD protein is only detected in immunoprecipitates from the membranefraction with serum from patient I₁₅ and I₁₆ (compare lanes 15-16 withlanes 17-18).

FIG. 1B depicts the immunoprecipitation of membrane fractions of³⁵S-methionine labeled islet cell proteins with sera from newlydiagnosed diabetic patients I₁₅ and I₆₂-I₈₀ (lanes 3-21) and healthycontrols C₁ and C₃₁-C₃₈. The diabetic sera recognize either the 38 kDprotein alone, the GAD₆₅ protein alone, both proteins or no specificprotein.

FIG. 2 is a fluorogram of an SDS-PAGE showing sequentialimmunoprecipitation of ³⁵S methionine labeled rat islets with serapositive for the anti-38 kD protein antibody and for the anti-GAD₆₅antibody. Lanes 1 and 4 show immunoprecipitation with an anti-GAD₆₅antibody positive stiff-man syndrome serum⁴ (Lane 1), and a diabeticpatient serum I₅ (lane 4). The supernatant after immunoprecipitation wasthen subjected to a second immunoprecipitation with anti-38 kD antibodypositive diabetic sera I₁₅ and I₄ (lanes 2 and 5). The supernatant afterimmunoprecipitation in lane 2 was then subjected to a thirdimmunoprecipitation with an anti-38 kD and anti-GAD₆₅ antibody positiveserum I₁₆ (lane 3). GAD₆₅ and the 38 kD protein were found not to affectthe immunoprecipitation of each other.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention results from our discovery that antibodies to aparticular 38 kD protein of pancreatic β-cells (which are theinsulin-secreting cells of the islets of Langerhans) are released intosera of a significant subpopulation of prediabetic human patients. Inparticular, it has been found that such antibodies appear to be presentin the sera of at least about 10% of prediabetic patients, withdetectable levels of the autoantibodies appearing at the early stages ofβ-cell destruction, usually at least one year prior to clinical onset ofinsulin dependent diabetes mellitus (IDDM) and often two years or moreprior to onset of the disease. Thus, the presence of autoantibodies tothe 38 kD autoantigen can serve as a useful marker for identifying thoseotherwise asymptomatic patients who are susceptible to developing IDDMat a later time. In particular, serum autoantibodies to the 3a kDautoantigen are useful for diabetes screening when combined withdetection of other serum markers, particularly autoantibodies to the 64kD pancreatic β-cell autoantigen (GAD₆₅).

The 38 kD autoantigen is a membrane-bound amphiphilic protein present inthe pancreatic islets or β-cells of mammalian species, such as human andrats, and is characterized by a pI in the range from about 5.4 to 6.1.The nominal molecular weight and pI range are based on comparisons withknown proteins, as described in detail in the Experimental section. Itwill be appreciated, of course, that the molecular weight and pI rangethus determined are subject to experimental error, and the presentinvention is not limited to these nominal characteristics of theprotein. Instead, the present invention relates to the detection ofautoantibodies against the autoantigen which has been isolated andidentified in FIGS. 1 and 2, as described hereinafter in theExperimental section. It is the discovery that these autoantibodies arepresent in patient sera which is critical to the present invention, notthe precise physical characteristics of the protein autoantigen.

The examples reported in the Experimental section rely on vigorousextraction of ³⁵S-methionine labeled rat islet cell proteins in adetergent. The 38 kD autoantigen protein of the present invention isrelatively insoluble in aqueous media, and it has previously beendifficult to obtain suitable cell extracts for use inimmunoprecipitation experiments. The particular extraction methodstaught in the Experimental section; however, have provided a reliablebasis for detecting presence of autoantibodies to the 38 kD autoantigen.Using the effectively solubilized islet cell proteins, it has been foundthat autoantibodies to the autoantigen are present in prediabeticpatients with a distribution different than that found forautoantibodies to the 64 kD autoantigen (GAD₆₅) who are positive forislet cell antibodies (ICA) by immunofluorescence. Thus, it has beendemonstrated that detection of autoantibodies to the 38 kD autoantigenin sera is a useful predictive marker for asymptomatic patients at riskof developing IDDM.

The methods of the present invention will be used to screen asymptomaticpatients to determine those patients who are prediabetic, i.e. at riskof developing IDDM in the future. By “asymptomatic,” it is meant thatthe patient is free from clinical symptoms of diabetes and has not yetsuffered sufficient damage to the insulin-producing β-cells to beclinically identified as having IDDM. By “prediabetic” it is meant thatthe patient has developed detectable levels of circulatingautoantibodies to an autoantigen on the pancreatic β-cells, such as the38 kD autoantigen, and is at significant risk of developing IDDM in thefuture. The clinical onset of IDDM may be determined by conventionalclinical indicia as well described in the medical literature.

A wide variety of suitable assay formats exist for detectingautoantibodies to the 38 kD autoantigen in accordance with theprinciples of the present invention. As described irk detail in theExperimental section hereinafter, the presence of autoantibody inpatient sera can be determined by immunoprecipitation with labeled 38 kDautoantigen. Labeled autoantigen can be obtained by growing suitablemammalian islet cells, such as rat or human islets, in the presence of alabeled amino acid precursor, such as ³⁵S-methionine. The resultinglabeled 38 kD antigen can then be extracted using the protocol describedin the Experimental section, or other equally vigorous procedures forsolubilizing the relatively insoluble autoantigen. The extractedautoantigen is then reacted with patient sera, and the resultingreaction products separated using conventional techniques, such asSDS-PAGE, or the like. Such immunoprecipitation protocols have theadvantage that no purification of the 38 kD autoantigen is required andthat identification of autoantibodies to the 64 kD autoantigen (GAD⁶⁵)can be performed simultaneously.

Generally, however, it will be desirable to utilize more convenientassay formats, such as immunoassays, enzyme assays, and the like. Suchimmunoassays and enzyme assays typically rely on exposing purified 38 kDautoantigen, or otter ligand capable of binding autoantibodies to the 38kD autoantigen, to a serum sample and detecting specific binding betweenthe ligand and autoantibodies for the 38 kD autoantigen which may bepresent in the serum. Binding between the autoantibodies and the 38 kDautoantigen or equivalent ligand indicates that the autoantibodies arepresent in the serum sample and is diagnostic of a prediabetic conditionin an asymptomatic patient prior to clinical onset of the disease.

The particular assay protocol chosen is not critical, and it isnecessary only that the assay be sufficiently sensitive to detect athreshold level of the autoantibodies. Such threshold level should be aslow as possible, with the only lower limit being that the assays mustdistinguish negative sera with no autoantibodies to the 38 kDautoantigen as present. It will be appreciated that in the very earlystages of β-cell destruction, very low levels of the autoantibodies maybe present. Thus, the presence of any autoantibodies above the negativebackground or control level will be diagnostic of the prediabeticcondition.

Suitable assays include both solid phase (heterogeneous) and non-solidphase (homogeneous) protocols. The assays may be run using competitiveor non-competitive formats, and using a wide variety of labels, such asradioisotopes, enzymes, fluorescers, chemiluminescers, spin labels, andthe like. A majority of suitable assays rely on heterogeneous protocolswhere the ligand is bound to a solid phase which is utilized to separatethe ligand-autoantibody complex which forms when autoantibody is presentin the serum sample. A particular advantage of using a purified ligandis that it facilitates the preparation of a solid phase for use in theassay. That is, the ligand may be conveniently immobilized on a varietyof solid phases, such as dipsticks, particulates, microspheres, magneticparticles, test tubes, microtiter wells, and the like.

The solid phase is exposed to the serum sample so that the autoantibody,if any, is captured by the ligand. By then removing the solid phase fromthe serum sample, the captured autoantibodies can be removed fromunbound autoantibodies and other contaminants in the serum sample. thecaptured autoantibody may then be detected using the non-competitive“sandwich” technique where labeled ligand for the autoantibody isexposed to the washed solid phase. Alternatively, competitive formatsrely on the prior introduction of soluble, labeled autoantibody to theserum sample so that labeled and unlabeled forms may compete for bindingto the solid phase. Such assay techniques are well known and welldescribed in both the patent and scientific literature. Exemplaryimmunoassays which are suitable for detecting the autoantibodies inserum include those described in U.S. Pat. Nos. 3,791,932; 3,817,837;3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876,the disclosures of which are incorporated herein by reference.

Particularly preferred are sensitive enzyme-linked immunosorbent assay(ELISA) methods which are described in detail in U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,879,262; and 4,034,074. Such ELISAassays can provide measurement of very low titers of the autoantibodies.

According to the preferred ELISA technique, the purified ligand is boundeither covalently or non-covalently to a solid surface. The solidsurface is exposed to the serum sample where autoantibody present in thesample is captured and bound. Typically, the ligand on the solid phasewill be present in excess so that the entire quantity of autoantibodymay be bound. After separating the solid phase and washing its surface,the solid phase can be exposed to labeled reagent capable ofspecifically binding the captured autoantibody. The labeled reagent maybe labeled purified ligand, or may be other ligand capable of binding tothe autoantibody, e.g., labeled anti-human antibody. In this way, labelis bound to the solid phase only if autoantibody was present in theserum sample. The enzyme labels may be detected by conventionalvisualization techniques, e.g., production of a colored dye,chemiluminescence, fluorescence, or the like.

A second preferred embodiment comprises radioimmunoassays (RIA) whichare performed using a solid phase which has been prepared as describedabove. The solid phase is exposed to the serum sample in the presence ofradiolabeled autoantibodies which can compete for binding to theimmobilized ligand. In this way, the amount of radiolabel bound to thesolid phase will be inversely proportional to the amount ofautoantibodies initially present in the serum sample. After separationof the solid phase, non-specifically bound radiolabel can be removed bywashing, and the amount of radiolabel bound to the solid phasedetermined. The amount of bound radiolabel, in turn, can be related tothe amount of autoantibodies initially present in the sample.

Methods according to the present invention will preferably combinedetection of autoantibodies to the 38 kD autoantigen with detection ofother known IDDM markers, such as autoantibodies to other β-cellautoantigens, particularly autoantibodies to the 64 kD autoantigen(GAD₆₅) and insulin autoantibodies, and most particularly autoantibodiesto the 64 kD autoantigen (GAD₆₅). It has been found that the presence ofautoantibodies to the 38 kD autoantigen is overlapping but notcoextensive with the presence of autoantibodies to the 64 kD autoantigenin prediabetic patient sera. Thus, presence of autoantibodies to eitheror both of the 38 kD autoantigen and. the 64 kD autoantigen (GAD₆₅) willbe diagnostic of the prediabetic condition.

Methods and compositions suitable for detecting autoantibodies to the 64kD autoantigen (GAD₆₅) in patient sera are described in detail incopending applications Ser. No. 07/756,207 (which is equivalent topublished PCT Application WO 92/04632) and 07/984,935, the disclosuresof which are both fully incorporated herein by reference. Detection ofautoantibodies to the 64 kD autoantigen (specifically the α and β formsof GAD₆₅) by immunoprecipitation is described in detail in theExperimental section hereinafter.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL

Methods

Neonatal rat islets were isolated and labeled with ³⁵S-methionine asdescribed in S. Baekkeskov et al., (1989) Diabetes, 38, 1133-1141.Islets were swollen on ice for 10 minutes in HEMAP buffer (10 mM Hepes,pH 7.4, 1 mM MgCl₂, 1 mM EGTA, 1 mM aminoethyl-isothiorunium bromidehydrobromide, and 0.2 mM pyridoxal phosphate), followed byhomogenization by 20 strokes in a glass homogenizer. The homogenate wascentrifuged at 100,000 g for 1 hour to obtain a cytosol and aparticulate membrane fraction. The particular membrane fraction wasextracted in HEMAP-buffer with 2% Triton X-114 for 2 hours by repeateddispersion through a bended pipette tip, followed by centrifugation at100,000 g to remove debris. Amphiphilic proteins in both the cytosolfraction and the membrane extract were purified by temperature-inducedTX-114 phase separation. See Baekkeskov et al., (1990) Nature347:151-156. The detergent phase of membrane or cytosol fractions wereprecleared with normal human serum before immunoprecipitation with theindicated sera, as described in Baekkeskov et al. (1989), supra.Extracts of 250 rat islets (FIGS. 1A and 1B) and 500 rat islets (FIG. 2)were used per immunoprecipitate. Immunoprecipitates were analyzed bySDS-PAGE using 15% gels and processed for fluorography, as described inBaekkeskov et al. (1989), supra.

The 38kD protein is an amphiphilic β-cell membrane protein of pI 5.4-6.1

The distribution of the insoluble 38 kD protein into cytosolic andparticulate islet cell fractions was analyzed, and cytosolic andmembrane proteins were subjected to a Triton X-114 phase separation toassess the amphiphilicity of the 38 kD protein (FIG. 2). In contrast tothe GAD autoantigen which is found as a soluble hydrophilic, a solubleamphiphilic, and a membrane bound amphiphilic form, as described inChristgau et al., (1992) J. Cell Biol., 118:309-320 and Christgau etal., (1991) J. Biol. Chem., 266:21257-21264, (FIG. 1A compare lanes 16and 18), the 38 kD protein was only detected in the particulatefraction, where it partitioned into the detergent phase (FIG. 1A, lanes15 and 16). Thus, the 38 kD protein is an amphiphilic membrane protein.

The 38 kD protein was detected as a broad band on fluorograms ofSDS-gels suggesting heterogeneity in size and/or charge (FIGS. 1 and 2).Two dimensional gel electrophoresis using isoelectric focusing in thefirst dimension and SDS-PAGE in the second dimension, as described inBaekkeskov et al. (1989) supra, revealed 7 spots of similar relativemolecular weight. The corresponding pI's of 5.4-6.1 were determined bycoelectrophoresis with carbamylated creatine phosphokinase charge chainmarkers and known HeLa cellular proteins.

In an analysis of neuroendocrine and non-endocrine cell lines, the 38 kDantigen was only detected in immunoprecipitates of βTC3 cells derivedfrom a transgenic mouse β-cell tumor, as described in Efrat et al.,Proc. Natl. Acad. Sci., (1988) 85:9037-9041, whereas glucagon producingαTC cells, as described in Powers et al., (1990) Diabetes 39:406-414, aswell as all other cell lines tested in this study were negative (Table1). Thus expression of the 38 kD protein appears to be restricted topancreatic β-cells.

TABLE 1: ANALYSIS OF EXPRESSION OF THE 38 kD PROTEIN IN DIFFERENT CELLLINES: CELL LINE ORIGIN EXPRESSION βTC3^(b) Mouse pancreatic insulinomapos αTC-2^(c) Mouse pancreatic glucagonoma neg PC12^(a) Rat adrenalpheochromocytoma neg CHO^(a) Chinese hamster ovary neg HeLa^(a) Humanovarian adenocarcinoma neg T₄₇D^(a) Human ductal breast carcinoma negSk-NEP-1^(a) Human nephroblastoma neg Cos-1^(a) Monkey kidney tumor negCV-1^(a) Precursor of Cos-1 neg HepG2^(a) Human hepatocellular carcinomaneg BHK-21^(a) Baby hamster kidney neg HTC^(d) Rat hepatoma negTERA-2^(a) Human teratocarcinoma neg CCD-118Sk^(a) Human fibroblast neg

Methods: Cell lines were cultured according to established methods andlabeled with ³⁵S-methionine, Baekkeskov et al. (1989) supra. Membraneextracts were prepared and immunoprecipitated as described for neonatalrat islets in Methods, using serum I₁₅ and serum C₁. Theimmunoprecipitates were analyzed by SDS-PAGE and fluorography,Baekkeskov et al. (1989) supra.

a: American Type Culture Collection, Bethesda, Md.

b: Efrat et al. (1988) Proc. Natl. Acad. Sci. 85:9037-9041.

c: Powers et al. (1990) Diabetes 39:406-414.

d: Nagata and Yoon (1992) Diabetes 41:998-1008.

The 38 kD protein is not a fragment of GAD

Antibodies in sera from type 1 diabetic patients recognize a 37 kDtrypsin fragment of GAD₆₅, as described in Christie et al., (1990) J.Exp. Med., 172:789-794, and Christie et al., (1992) Diabetes,41:782-878. Such sera do not always recognize the full length native GADmolecule, suggesting that a sequestered epitope may become exposed upontrypsinization. In light of this report, we have addressed the questionwhether the 38 kD protein is a fragment of GAD or otherwise related toGAD.

Islet cell membrane extracts were sequentially immunoprecipitated withsera that contained anti-GAD₆₅ or anti-38 kD antibodies. Supernatantsdepleted for immunoreactive, GAD₆₅. were still positive for the 38 kDprotein (FIG. 2). Similarly supernatants depleted for immunoreactive 38kD protein still contained the GAD₆₅ molecule. Thus, the native 38 kDprotein and the native GAD₆₅ molecule do not display immunologicalcrossreactivity. Next we analyzed whether anti-38 kD sera recognize a37/40 kD fragment of GAD₆₅, which was generated by a mild trypsindigestion of either protein. Although several anti-GAD sera recognizethe 37/40 kD GAD₆₅ trypsin fragment, the single anti-38 kD antibodypositive sera tested were negative. Finally two 37 kD antibody positivesera did not immunoprecipitate the 38 kD antigen. These resultsdemonstrate that the 38 kD protein is distinct from GAD.

Autoantibodies to the 38 kD antigen are present in a subgroup ofprediabetic and diabetic individuals and complement GAD autoantibodies

The incidence of 38 kD antibodies was analyzed and compared withprevalence of GAD antibodies and ICA in three groups. (FIG. 1 and Table2): (i) 37 children who developed diabetes ≦5 years of age and 38controls in the same age group, (ii) 49 individuals who developeddiabetes at >5 years and 25 controls in the same age group and (iii) 44individuals (age 2.6-49.9 years at clinical onset) whose sera had beenobtained prior to clinical onset of type 1 diabetes.

The results from the three groups are shown in Table 2. In a total of130 patients, who were analyzed either in the prediabetic period or atthe clinical onset of disease, 22 (17%) were anti-38 kD antibodypositive compared to 100 (77%), who were anti-GAD₆₅ antibody positive.Six patients were positive for anti-38 kD antibodies only, whereas 16had both anti-38 kD and anti-GAD₆₅ antibodies. Thus 106 (82%) werepositive for antibodies to either or both antigens. Anti-38 kD as wellas anti-GAD₆₅ antibodies were detected at clinical onset in children whodeveloped type 1 diabetes as early as 1.3 and 0.8 years of agerespectively. Since the duration of β-cell autoimmunity in those veryyoung children can only have been short, both proteins are likelytargets of the primary rather than secondary autoimmune processesdirected to the β-cell in the human disease. This circumstance issupported by the appearance of antibodies to both antigens several yearsbefore the clinical onset of type 1 diabetes. Thus the six anti-38 kDantibody positive individuals in the prediabetic group were all positivein the very first sample available (3, 9, 25, 33, 53, and 74 monthsbefore clinical onset, respectively), and antibodies persisted infollow-up samples which were available from the prediabetic period in3/6 patients. Similarly anti-GAD₆₅ antibodies were detected in samplesobtained 3-85 months before clinical onset, a result consistent with ourearlier studies, as described in Baekkeskov et al. J. Clin. Invest.,79(3), 926-934 (1987), and Atkinson et al., Lancet, 335:1357-1360(1990). Thus both anti-38 kD and anti-GAD₆₅ antibodies can be detectedin sera up to several years before clinical onset indicating that theyare markers of early β-cell destruction.

TABLE 2 INCIDENCE OF AUTOANTIBODIES To A 38 kD β-CELL NEMBRANE PROTEININ TYPE 1 DIABETES AND COMPARISON WITH GAD₆₅ab AND ICA Avg. age atdiagnosis or at Incidence sampling of Incidence 38 kD sera 38 kDIncidence and/or (controls) Range auto- GAD₆₅ auto- GAD₆₅ auto- GROUP nyears years F/M ICA^(a) antibodies antibodies antibodies Newly diagnosed37 2.9 ± 1.4 0.8-5.0 15/22 32^(b)/37 4^(c)/37 30/37 30/37 diabeticpatients (68%) (86%) (11%) (81%) (81%) <5 years of age Healthy controls38 2.8 ± 1.3 0.9-5.0 18/20 0/38 0/38 0/38 0/38 <5 years of age (90%)(0%) (0%) (0%) (0%) Newly diagnosed 49 13.1 ± 9.22 5.1-57.0 20/2938^(c)/49 12^(f)/49 37/49 41/49 diabetic patients (69%) 77% (24%) (76%)(84%) >5 years of age Healthy controls 25 14.9 ± 12.6 5.1-54.2 10/150/25 0/25 0/25 0/25 >5 years of age (68%) (0%) (0%) (0%) Prediabetic 4419.2 ± 12.5 2.6-49.9 14/30 28^(d)/44 6^(g)/44 33/44 35/44 Individuals(47%) (64%) (14%) (75%) (80%) 3-85 mo. before clinical onset ^(a)ICA wasanalyzed by indirect immunofluorescence on frozen sections of humanpancreas, as described in Greenbaum et al., (1992) Diabetes,41:1570-1574. ^(b)Four of whom were both anti-GAD₆₅ and anti-38 kDantibody negative. ^(c)Six of whom were both anti-GAD₆₅ and anti-38 kDantibody negative. ^(d)None of whom were anti-GAD₆₅ and anti-38 kDantibody negative. ^(e)All of whom also had GAD₆₅ autoantibodies.^(f)Eight of whom also had GAD₆₅ autoantibodies. ^(g)Four of whom alsohad GAD₆₅ autoantibodies. ^(n)Number of patients in Group.

The incidence of ICA detected by immunofluorescence of frozen sectionsof human pancreas was 75% (98/130). In the three groups, the anti-38 kDand/or anti-GAD65 immunoprecipitation assays detected a total of 18individuals which were negative for ICA by the immunofluorescence assayindicative of a lower sensitivity of the latter method to detectantibodies to those antigens. Blocking experiments have shown that theICA response can progress from GAD-restricted to non-GAD restrictedduring the prediabetic period in some individuals which suggests aspreading of antigen reactivity during prolonged periods of .beta.-celldestruction, as described in Atkinson et al., (1993) J. Clin. Invest.91:350-356. Interestingly, all ICA positive individuals in theprediabetic group were positive for either anti-38 kD antibodies,anti-GAD₆₅ antibodies or both. In contrast 4 and 6 ICA positiveindividuals in the young and older newly diagnosed groups respectivelywere negative for both anti-38 kD and anti-GAD₆₅ antibodies, suggestingthat the humoral autoimmune response in those individuals may includeother target molecules at the time of clinical onset.Immunoprecipitations did not reveal islet cell protein(s) that werespecifically recognized by those sera (results not shown). It isconceivable that ICA reactivity in those anti-38 kD and anti-GAD₆₅antibody negative sera may be directed to non-protein molecules likegangliosides, as described in Nayak et al., (1985) Diabetes, 34:617-619.Finally, 8 individuals in the prediabetic group and 2 and 3 individualsin the young and older newly diagnosed groups respectively were negativefor antibodies by all three assays.

The anti-38 kD antibodies analyzed in this study recognized their targetunder native but not denaturing conditions, suggesting that anti-38 kDantibodies, much as anti-GAD₆₅ antibodies, are primarily directed towardconformational epitopes. Although the destruction of β-cells is believedto be mediated primarily by T-cells rather than antibodies andcomplement, islet cell antibodies present during early phases of β-celldestruction seem likely to be directed toward the same antigens as thepathogenic T-cells. We have extensively analyzed immunoprecipitates ofdetergent lysates of ³⁵S-methionine labeled islets with diabetic andcontrol sera by one and two dimensional gel-electrophoresis in attemptsto detect additional islet cell proteins that are specificallyrecognized by autoantibodies in type 1 diabetes. The stringentconditions of immunoprecipitation require that antibodies must (1) be ofthe IgG isotype (for binding to Protein A-Sepharose®), and (2) be ofsufficient affinity and specificity to recognize their target protein inthe midst of an abundance of other islet cell proteins. Using thisassay, we have been unable to detect proteins other than GAD and the 38kD protein that are consistently and specifically recognized by asignificant fraction of diabetic sera. In particular, insulin, and a 69kD protein with homology to bovine serum albumin, reported elsewhere tobe targets of antibodies in diabetic sera, as described in Palmer etal., (1983) Science, 222:1337-1339 and Karjalainen et al., (1992) N.Eng. J. Med. 327:302-307, were not detected in immunoprecipitates,Baekkeskov et al. (1989), supra. Thus, strong IgG responses to proteinantigens may be limited to the GAD₆₅ and 38 kD molecules in the humandisease. Since high titer anti-GAD₆₅ and anti-38 kD IgG antibodies aredetected in the early phases of β-cell autoimmunity, we predict thepresence of activated CD4⁺ T-helper cells recognizing each of thosemolecules. In fact, reactivity to both GAD and to a 38 kD β-cell proteinhas been demonstrated using T-cell lines from newly diagnosed diabeticpatients, as described in Atkinson et al., (1992) Lancet, 339:458-459;Diaz et al., (1992) Diabetes, 41:118-121; Roep et al., (1990) Nature,345:632-634; and Roep et al., (1991) Lancet, 337:1439-1441. The relationof the 38 kD antigen described herein with that 38 kD T-cell stimulatoryprotein, Roep et al., (1990) supra and Roep et al., (1991), supra,remains to be clarified.

Although the foregoing invention has been described in some detail byway of illustration and example, for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for assessing the risk of developinginsulin dependent diabetes mellitus (IDDM) in an asymptomatic humanpatient, said method comprising; contacting a serum sample from apatient with a ligand characterized in its natural state by amembrane-bound islet cell location, a molecular weight of 38 kD, anamphiphilic charge, and a pI in the range from 5.4 to 6.1 thatspecifically binds to autoantibodies to the ligand assaying specificbinding of the ligand to the sample; wherein specific binding of theligand to the sample indicates the presence of auatoantibodies to theligand in the serum sample from the patient, and thereby a likelihood ofthe patient developing IDDM.
 2. A method as in claim 1, wherein theautoantibodies are detected by immunoprecipitation with labeled ligand.3. A method as in claim 1, wherein the autoantibodies are detected byreaction with the ligand immobilized on a solid phase, separation of thesolid phase from the serum sample, and detection of autoantibodies boundto the solid phase.
 4. A method of detecting autoantibodies to a 38 kDautoantigen, wherein the 38 kD autoantigen is an anphiphilicmembrane-bound islet cell protein having a pI in the range from 5.4 to6.1, the method comprising: contacting a serum sample from a patientwith a ligand characterized in its natural state by a membrane-boundislet cell location, a molecular weight of 38 kD, an amphiphilic charge,and a pI in the range from 5.4 to 6.1 assaying specific binding of theligand to the sample; wherein specific binding of the ligand to thesample indicates the presence of autoantibodies to the 38 kD autoantigenin the serum sample from the patient.